Rhodococcus strain DP-4 and its application in degrading decaline

Abstract

The present application belongs to the field of biological bacterial agent and organic pollutant remediation technology, and particularly relates to a strain of Rhodococcus DP-4 and application thereof in degrading Dechlorane Plus (DP). The present application first separates and identifies a strain of Rhodococcus DP-4 capable of degrading DP from exogenous DP contaminated soil, filling the research blank of existing DP degrading bacterial strain resources. Research shows that the Rhodococcus can utilize DP as the sole carbon source and energy to achieve its biodegradation, and the degradation rate presents a change trend of first increasing and then tending to be flat as the culture time prolongs. In addition, in the present application, the Rhodococcus DP-4 has excellent environmental adaptability when degrading DP, the temperature is 20-40 DEG C, the pH is 6.0-9.0, and the initial concentration of DP is 1-50 mg / L, which can be effectively utilized by the Rhodococcus DP-4, and has good practical application potential and development value.

Description

Technical Field

[0001] This invention belongs to the field of biological agents and organic pollutant remediation technology, specifically relating to a strain of Rhodococcus DP-4 and its application in degradation and cloning. Background Technology

[0002] Declone (DP) is a synthetic chlorine-containing flame retardant. Due to its excellent compatibility and stable physicochemical properties, it is widely used in industrial manufacturing fields such as protective coatings for wires and cables, nylon polymer materials, precision electronic components, and various electronic and electrical appliance housings. It is an indispensable functional additive in industrial production. In the early 21st century, persistent organic pollutants (PAPs) were officially included in the Stockholm Convention's control list, completely banning their production and application globally. Against this backdrop of industry transformation, DP, with its outstanding advantages such as low production cost, stable chemical structure, and strong weather resistance, was identified by the European Union as a core alternative flame retardant chemical to PAPs. Market demand surged, and industrial production and application scale experienced explosive growth.

[0003] Large-scale and widespread continuous use allows DP to be continuously released into the natural environment through various pathways such as material wear, media dissolution, and product disposal, resulting in the continuous enrichment and accumulation of DP in the environmental system. Numerous studies have confirmed that DP perfectly matches the core characteristics of persistent organic pollutants (POPs), possessing extremely strong environmental persistence, hydrophobicity and lipophilicity, bioaccumulation, and long-distance environmental migration capabilities. It can remain in natural media such as soil and water bodies for a long time, and can be enriched and amplified step by step through the food chain, posing a potential and persistent threat to the balance of terrestrial and aquatic ecosystems and human health.

[0004] As a novel persistent organic pollutant, our understanding of the environmental occurrence of diest pollutants (DP) is relatively recent. In 2006, researchers first detected DP in the sedimentary environment and aquatic organisms of the Great Lakes in North America, thus initiating research on DP environmental pollution. Subsequent global environmental monitoring data show that DP is now widely distributed in various natural environmental media and biological samples worldwide, and its global pollution characteristics, ecological risks, and health hazards have gradually become a research hotspot in environmental science. Given DP's widespread distribution, long-term persistence, and hidden risks, developing efficient, green, and low-cost DP pollution remediation technologies is of significant practical value and urgent research importance for controlling regional environmental pollution, maintaining ecological security, and protecting human health.

[0005] Currently, the exploration of remediation technologies for DP-contaminated environments both domestically and internationally is still in the early stages of research and development. A mature and scalable remediation system has not yet been established. Existing remediation methods mainly focus on two major technical directions: physicochemical remediation and phytoremediation, which can initially reduce and remove DP pollutants from the environment. However, existing mainstream technologies have significant application shortcomings, greatly limiting their practical engineering application: physicochemical remediation technologies have relatively high process maturity, but generally suffer from high operation and maintenance costs, complex treatment processes, and a tendency to cause secondary environmental pollution; phytoremediation technologies are green and environmentally friendly, with no secondary pollution, but are constrained by factors such as plant growth cycles, bioaccumulation and degradation capabilities, and environmental adaptability, resulting in low pollutant degradation efficiency, long remediation cycles, and unstable treatment effects.

[0006] Compared to traditional remediation methods, microbial degradation technology, with its unique advantages such as being green and low-carbon, low-cost, effective in-situ remediation, and free from secondary pollution, is widely recognized by the industry as the core technology with the greatest potential for industrial application in the treatment of organic pollution. Basic research data from Hooker Chemical Company (now OxyChem), a company that develops DP (polychlorinated biphenyls), confirms that microorganisms can degrade and transform DP under aerobic conditions, providing a theoretical basis for the development of microbial remediation technology. Currently, research on microbial degradation and remediation technologies for traditional classic POPs such as organochlorine pesticides and polychlorinated biphenyls (PCBs) has formed a complete theoretical system and technological achievements both domestically and internationally. However, research on the microbial remediation of emerging pollutants such as DP remains relatively scarce, with few reports on related mechanisms of action, functional strains, and degradation processes. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Rhodococcus DP-4 and its application in degradation and cloning. The Rhodococcus DP-4 can efficiently degrade DP and has advantages such as high degradation efficiency and short treatment cycle.

[0008] This invention provides a strain of Rhodococcus DP-4, with accession number CGMCC No.38358.

[0009] The present invention also provides a bacterial agent containing Rhodococcus DP-4 as described in the above technical solution.

[0010] Preferably, the OD of Rhodococcus DP-4 in the bacterial agent is... 600 It is 1.5~2.0.

[0011] Preferably, the active ingredient of the bacterial agent includes a bacterial suspension of Rhodococcus DP-4.

[0012] The present invention also provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating Rhodococcus DP-4 into a liquid culture medium for amplification culture to obtain the bacterial agent.

[0013] Preferably, the amplification culture conditions include: a temperature of 20~35℃, a time of 18~24 h, and a rotation speed of 150~180 r / min.

[0014] The present invention also provides a method for degrading Declone, comprising the following steps: applying the bacterial agent described in the above technical solution to a Declone contaminated sample or environment for degradation treatment.

[0015] Preferably, the degradation treatment is carried out at a temperature of 20-40°C and a pH value of 6.0-9.0.

[0016] Preferably, the concentration of the declone is 1~50 mg / L.

[0017] The present invention also provides the application of Rhodococcus DP-4 as described in the above technical solution, or the bacterial agent as described in the above technical solution, or the bacterial agent prepared by the preparation method described in the above technical solution, or the method described in the above technical solution, in the bioremediation of Declone contaminated media or environment.

[0018] Beneficial effects: This invention marks the first time that a *Rhodococcus DP* strain, DP-4, capable of degrading DP, has been isolated and identified from exogenously DP-contaminated soil, filling a research gap in existing DP-degrading bacterial resources. Studies show that this *Rhodococcus DP* strain can utilize DP as its sole carbon and energy source for biodegradation, and the degradation rate exhibits an overall trend of first increasing and then leveling off with prolonged culture time. Furthermore, in this invention, *Rhodococcus DP-4* demonstrates excellent environmental adaptability in DP degradation; it can be effectively utilized at temperatures ranging from 20 to 40°C, pH values ​​from 6.0 to 9.0, and initial DP concentrations from 1 to 50 mg / L, demonstrating significant practical application potential and development value.

[0019] Biological Preservation Instructions Rhodococcus DP-4, biologically classified as Rhodococcus sp. was deposited on April 22, 2026 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38358. The depository address is Building 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 The results of the plate streak of DP-4, the highly efficient DP-degrading bacterium in Example 1; Figure 2This is an experiment to determine the degradation effect of DP by Rhodococcus DP-4 in Example 2; Figure 3 This is a graph showing the effect of different temperatures on the DP degradation characteristics of Rhodococcus DP-4 in Example 3; Figure 4 This is a graph showing the effect of different pH values ​​on the DP degradation characteristics of Rhodococcus DP-4 in Example 3; Figure 5 The figure shows the effect of different initial DP concentrations on the DP degradation characteristics of Rhodococcus DP-4 in Example 3. Detailed Implementation

[0022] This invention provides a strain of Rhodococcus DP-4, with accession number CGMCC No.38358.

[0023] This invention is the first to enrich, purify, and screen DP-contaminated soil, obtaining a highly efficient DP-degrading Rhodococcus DP-4 strain. Rhodococcus DP-4 can utilize DP as its sole carbon and energy source for growth, reproduction, and efficient degradation, exhibiting advantages such as high degradation efficiency and short treatment cycle. It fills a research gap in existing DP-degrading bacterial strains and can provide novel microbial germplasm resources for the bioremediation of DP-contaminated environmental media.

[0024] The present invention also provides a bacterial agent containing Rhodococcus DP-4 as described in the above technical solution.

[0025] As one embodiment, the OD of Rhodococcus DP-4 in the bacterial agent of the present invention... 600 The value is 1.5 to 2.0, and more specifically, it can be 2.0. As one embodiment, the effective component of the bacterial agent of the present invention can be a bacterial suspension of Rhodococcus DP-4.

[0026] The present invention also provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps: inoculating Rhodococcus DP-4 into a liquid culture medium for amplification culture to obtain the bacterial agent.

[0027] As one embodiment, the amplification culture temperature of the present invention can be 20~35℃, and more preferably 30℃; the amplification culture time can be 18~24 h, and more preferably 24 h; the amplification culture rotation speed can be 150~180 r / min, and more preferably 150 r / min.

[0028] The present invention also provides a method for degrading Declone, comprising the following steps: applying the bacterial agent described in the above technical solution to a Declone contaminated sample or environment for degradation treatment.

[0029] In one embodiment, the degradation treatment temperature of the present invention can be 20~40℃, more preferably 25~35℃. In another embodiment, the pH value of the degradation treatment of the present invention can be 6.0~9.0, more preferably 7.0~8.0, and also preferably 7.0~7.5. In another embodiment, the initial concentration of the clone obtained in the present invention can be 1~50 mg / L, more preferably 1~10 mg / L. In another embodiment, the Rhodococcus DP-4 of the present invention can biodegrade DP using DP as the sole carbon and energy source, and the degradation rate generally shows a trend of first increasing and then leveling off over time; studies show that the degradation rate of DP by the Rhodococcus DP-4 gradually slows down after 5 days of culture, and the degradation rate tends to stabilize; after 7 days of culture, the degradation rate of DP at an initial concentration of 10 mg / L by this strain reaches 68.48%.

[0030] The present invention also provides the application of Rhodococcus DP-4 as described in the above technical solution, or the bacterial agent as described in the above technical solution, or the bacterial agent prepared by the preparation method described in the above technical solution, or the method described in the above technical solution, in the bioremediation of Declone contaminated media or environment.

[0031] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] The test samples and culture media used in the following examples are as follows: Test Samples: Soil samples were collected in 2024 along the North Canal of Shenyang University. The top 20 cm soil was collected using a multi-point mixing method. Plant roots, gravel, and other residues were removed, and the samples were refrigerated and transported back to the laboratory. After adding 10 mg / L of DP exogenously and aging for one month, the test soil with a DP content of 10.05 mg / kg was obtained.

[0033] Test culture medium: LB medium: 10.0 g sodium chloride, 10.0 g peptone, 5.0 g yeast extract, 1 L deionized water, adjust pH to 7.0, autoclave at 121℃ for 20 min.

[0034] Inorganic salt basal culture medium: sodium nitrate 4.0 g, potassium dihydrogen phosphate 1.5 g, sodium monohydrogen phosphate 0.5 g, ferric chloride 0.005 g, calcium chloride 0.01 g, magnesium sulfate 0.2 g, distilled water 1000 mL, adjust pH to 7, autoclave at 121℃ for 20 min.

[0035] Example 1 The isolation, identification, and degradation performance of Rhodococcus DP-4 were determined by the following steps: 1. Isolation of DP-degrading bacteria (1) Enrichment culture of DP-degrading bacteria Two g of fresh DP-aged soil sample was weighed and placed in 100 mL of inorganic salt medium. DP was added as the sole carbon source to a final concentration of 10 mg / L. The sample was cultured in a shaker at 30℃ and 150 r / min for 7 days to promote the initial growth of degrading bacteria under low DP concentration conditions. After the culture medium became turbid, 5 mL was transferred to 100 mL of fresh inorganic salt liquid medium, while the DP concentration was gradually increased to 20 mg / L. Subsequently, three similar transfers were performed, each time gradually increasing the DP concentration in fresh medium to 30, 40, and 50 mg / L. When the final DP concentration in the medium reached 50 mg / L, the initial enrichment culture was achieved.

[0036] (2) Isolation and purification of DP-degrading bacteria 1 mL of the final enriched culture was serially diluted to reduce interference from overly dense colonies. The diluted solution was evenly spread onto a solid inorganic salt medium plate with DP as the sole carbon source. First, the plate was spread evenly using a spreading stick, then single colonies were isolated using the streak plate method. The plate was inverted and incubated at 30°C, observed every 24 hours, until clearly visible single colonies appeared. Then, single colonies with good growth and differences in morphology and color were selected from the plate, labeled, and transferred to a new plate with DP as the sole carbon source for further isolation and purification. After repeated streak isolation, pure strains with consistent colony characteristics were obtained. Finally, four DP-degrading bacteria were screened and isolated (labeled DP-1, DP-2, DP-3, and DP-4).

[0037] 2. Screening of DP-efficient degrading bacteria (1) Preparation of bacterial suspension Single colonies of the four DP-degrading bacteria (DP-1, DP-2, DP-3, and DP-4) obtained in step 1 were picked and inoculated into LB liquid medium. The bacterial suspension was cultured at 30℃ and 160 r / min for 24 h with shaking. After reaching the logarithmic growth phase, the suspension was centrifuged at 8000 r / min for 5 min, and the supernatant was discarded. The bacterial cells were washed with sterile water and centrifuged again to collect the wet cells. This washing and centrifugation step was repeated twice. Finally, sterile water was added to adjust the bacterial suspension concentration, and the suspension was mixed by vortexing to achieve the desired OD value. 600 Reached version 2.0.

[0038] (2) Degradation performance experiment of DP-degrading bacteria Add an appropriate amount of DP-acetone mixture to an Erlenmeyer flask containing 20 mL of inorganic salt basal liquid culture medium, adjusting the final DP concentration to 10 mg / L; then add 10% ( V / V Inoculation amounts were respectively connected to OD 600 Bacterial suspensions of DP-1, DP-2, DP-3, and DP-4 with a pH of 2.0 were cultured at 30℃ and 150 r / min in the dark with shaking for 7 days. The residual DP concentration in the samples was measured after the culture. The treatment group inoculated with inactivated bacterial suspension served as a blank control, and all treatments were replicated in triplicate. The DP degradation ability of each strain is shown in Table 1.

[0039] Table 1. Degradation performance of different strains of DP

[0040] Table 1 shows that after 7 days of culture, strains DP-1, DP-2, DP-3, and DP-4 all possessed certain DP degradation capabilities, but the degradation capabilities varied significantly among different strains. Strain DP-1 exhibited the lowest degradation effect, with a degradation rate of only 15.12%; strains DP-2 and DP-3 showed moderate degradation capabilities, with degradation rates of 32.11% and 44.39%, respectively; strain DP-4 demonstrated the best degradation performance, achieving a DP degradation rate of 62.80%. Based on these results, DP-4 was selected as the dominant and highly efficient DP-degrading strain for further identification and related research.

[0041] 3. Identification of DP-4 species, which are highly efficient DP-degrading bacteria (1) Identification of the morphology and physiological and biochemical levels of the highly efficient degrading bacteria DP-4 Results of streak plating on LB medium ( Figure 1 As can be seen, DP-4 colonies are arranged in a relatively regular linear pattern, light orange in color, with clear and relatively flat edges, a slightly raised surface, and a relatively dense colony structure, making them easy to pick up. Following the methods described in the *Handbook of Systematic Identification of Common Bacteria*, the physiological and biochemical characteristics of the strain were determined. The results showed that the strain was positive for catalase, aerobic test, urea hydrolysis, and starch hydrolysis; negative for oxidase, methyl red, indole, and VP tests; and positive for Gram staining.

[0042] (2) Identification of 16S rDNA of DP-4, a highly efficient DP-degrading bacterium Genomic DNA of DP-4, a highly efficient DP-degrading bacterium, was obtained using the Ezup column-based bacterial genomic DNA extraction kit. The 16S rDNA gene of the bacteria was amplified by PCR and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis. The upstream primer sequence used for amplifying the 16S rDNA gene was as follows:

[0043] The NCBI database BLAST tool was used to perform homology comparison between the sequenced data and the GenBank database. The results showed that the sequence of this strain is similar to that of Rhodococcus spp. ( Rhodococcus The homology similarity between the two is 100%, and they are most closely related.

[0044] Based on the combined physiological and biochemical characteristics of the strain and the 16S rDNA sequence identification results, the strain was determined to belong to the genus Rhodococcus and named strain DP-4.

[0045] Example 2 The experiment to determine the degradation effect of DP by Rhodococcus DP-4 was conducted as follows: Add an appropriate amount of DP-acetone mixture to an Erlenmeyer flask containing 20 mL of inorganic salt basal liquid culture medium to bring the final DP concentration to 10 mg / L; then add 10% ( V / V The inoculation volume was connected to the OD. 600 DP-4 bacterial suspension with a pH of 2.0 was cultured at 30℃ and 150 r / min in the dark with shaking. Samples were taken on days 1, 3, 5, 7, and 9 of culture to determine the residual DP content in the culture medium. The treatment group inoculated with inactivated Rhodococcus DP-4 bacterial suspension served as a blank control. All experimental treatments were performed in triplicate. The test results are as follows: Figure 2 As shown.

[0046] Depend on Figure 2 It is evident that strain DP-4 can utilize DP as its sole carbon and energy source for biodegradation. The degradation rate initially increases and then levels off with prolonged culture time. On day 1 of culture, the degradation rate of DP by strain DP-4 was 41.36%. As culture time increased, the degradation rate continued to rise, reaching 66.27% on day 5, after which the degradation rate slowed and gradually stabilized. By day 7, the degradation rate of DP by strain DP-4 was 68.48%. In contrast, the DP loss rate of the inactivated bacterial suspension control group was only 4.56% after 9 days of shaker culture, indicating that abiotic pathways have limited effect on DP removal, and DP degradation is primarily driven by the biochemical metabolic processes of strain DP-4.

[0047] Example 3 Effects of different environmental factors on the degradation characteristics of Rhodococcus DP-4 1. Effects of different temperatures on the degradation characteristics of DP by Rhodococcus DP-4 Rhodococcus DP-4 was inoculated into LB liquid medium and cultured at 30℃ and 150 r / min for 24 h in a constant temperature shaker. The OD of the bacterial culture was then adjusted. 600 Value up to 2.0. At 10% ( V / VThe activated bacterial suspension was transferred to a basic inorganic salt culture medium at pH 7.0 with an initial DP concentration of 10 mg / L. The medium was cultured at five temperature gradients (20, 25, 30, 35, and 40 °C) under shaking at 150 r / min. The residual DP content in the culture medium was measured after 7 days. A treatment group inoculated with inactivated Rhodococcus DP-4 bacterial suspension served as a blank control. All experimental treatments were replicated in triplicate. Results are as follows: Figure 3 As shown.

[0048] Depend on Figure 3 The results showed that the culture temperature had a significant impact on the degradation characteristics of DP by Rhodococcus DP-4. This strain could degrade DP in the range of 20-40℃, with 25-35℃ being the optimal degradation temperature range. As the culture temperature increased, the DP degradation rate generally showed a trend of first increasing and then decreasing, with the best degradation effect at 25℃, reaching a degradation rate of 65.78%.

[0049] 2. Effects of different pH values ​​on the DP degradation characteristics of Rhodococcus DP-4 Rhodococcus DP-4 was inoculated into LB liquid medium and cultured at 30℃ and 150 r / min for 24 h with constant temperature shaking. The OD of the bacterial culture was then adjusted. 600 Value up to 2.0. At 10% ( V / V The activated bacterial suspension was transferred to basal inorganic salt culture media with an initial DP concentration of 10 mg / L and pH gradients of 6.0, 7.0, 7.5, 8.0, and 9.0, respectively, and cultured at 30℃ and 150 r / min for 7 days with shaking. The residual DP content in the culture medium was then measured. The treatment group inoculated with inactivated Rhodococcus DP-4 bacterial suspension served as a blank control. All experimental treatments were performed in triplicate. The results are as follows: Figure 4 As shown.

[0050] Depend on Figure 4 The results showed that pH had a significant impact on the degradation characteristics of DP by Rhodococcus DP-4. This strain could effectively degrade DP in the pH range of 6–9, with 7–7.5 being the optimal pH range for degradation. As pH increased, the DP degradation rate generally showed a trend of first increasing and then decreasing, reaching its highest level of 68.48% at pH 7.

[0051] 3. Effects of different initial DP concentrations on the DP degradation characteristics of Rhodococcus pyogenes DP-4 Rhodococcus DP-4 was inoculated into LB liquid medium and cultured at 30℃ and 150 r / min for 24 h with constant temperature shaking. The OD of the bacterial culture was then adjusted. 600 Value up to 2.0. At 10% ( V / VThe activated bacterial suspension was transferred to basic inorganic salt culture medium at pH 7.0 with initial DP concentrations of 1, 5, 10, 30, and 50 mg / L, respectively. The cultures were incubated at 30℃ and 150 r / min for 7 days with shaking. The residual DP content in the culture medium was then measured. The treatment group inoculated with inactivated Rhodococcus DP-4 bacterial suspension served as a blank control. All experimental treatments were performed in triplicate. The results are as follows: Figure 5 As shown.

[0052] Depend on Figure 5 It was found that different initial DP concentrations significantly affected the DP degradation characteristics of Rhodococcus DP-4. This strain could effectively degrade DP within an initial DP concentration range of 1–50 mg / L. Furthermore, with increasing initial DP concentration, the DP degradation rate generally showed a trend of first decreasing, then increasing, and then decreasing again. The strain exhibited the best degradation effect at an initial DP concentration of 1 mg / L, achieving a degradation rate of 71.43%. Rhodococcus DP-4 showed the optimal DP degradation performance under the conditions of a culture temperature of 25℃, a system pH of 7, and an initial DP concentration of 1 mg / L.

[0053] The above examples show that Rhodococcus DP-4 has high degradation efficiency for DP and a wide range of degradation conditions, which has great application advantages and value.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Rhodococcus ( Rhodococcus sp.) DP-4, accession number CGMCC No.38358.

2. An inoculum containing the Rhodococcus DP-4 of claim 1.

3. The microbial agent according to claim 2, characterized in that, The OD of Rhodococcus DP-4 in the bacterial agent 600 It is 1.5~2.

0.

4. The microbial agent according to claim 2 or 3, characterized in that, The active ingredient of the bacterial agent includes a suspension of Rhodococcus DP-4.

5. The method for preparing the microbial agent according to any one of claims 2 to 4, characterized in that, The process includes the following steps: inoculating Rhodococcus DP-4 into a liquid culture medium for amplification culture to obtain the bacterial agent.

6. The preparation method according to claim 5, characterized in that, The amplification culture conditions include: a temperature of 20~35℃, a time of 18~24 h, and a rotation speed of 150~180 r / min.

7. A method for obtaining clones through degradation, characterized in that, The process includes the following steps: applying the bacterial agent according to any one of claims 2 to 4 or the bacterial agent prepared by the preparation method shown in claim 5 or 6 to the clone-contaminated sample or the environment for degradation treatment.

8. The method according to claim 7, characterized in that, The degradation treatment is carried out at a temperature of 20-40℃ and a pH value of 6.0-9.

0.

9. The method according to claim 7 or 8, characterized in that, The concentration of the declon is 1~50 mg / L.

10. The application of the Rhodococcus DP-4 of claim 1, or the bacterial agent of any one of claims 2 to 4, or the bacterial agent prepared by the preparation method of claim 5 or 6, or the method of any one of claims 7 to 9, in the bioremediation of Decloal contaminated media or environment.

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